Cell motility is governed by the interaction between cytoskeletal proteins and other proteins embedded in the cell membranes. Cytoskeletal proteins which partake in the generation of force within the cell are termed contractile proteins. The energy source of such force generating activity is ATP.
Two predominant contractile proteins in all animal cells are actin and myosin. Actin is present in both soluble and polymerized forms. For example, filamentous (polymerized) actin interacts with myosin to contract or relax muscle tissues, to transport cell organelles through the intracellular medium, to cause cell movement, and to separate daughter nuclei during cytokinesis.
Myosin has a rodlike structure composed of heavy chain and light chain isoforms. Myosin light chains (MLCs) are associated stoichiometrically with the globular N-terminal domain of myosin heavy chains (MHCs). The globular domain also contains the ATP-binding and actin-binding sites. The MHC C-terminal domain structure is predominantly in the form of an .alpha.-helical coil, which interacts with the C-terminal domain of a second MHC monomer to form a coiled coil higher order structure. Heavy-chain isoforms appear to be present in all tissue types studied and may regulate maximum shortening velocity of the myofibrils and alter the sensitivity of actinomyosin to intracellular calcium ion concentrations. Two MHC isoforms expressed in smooth muscle are derived from alternate splicing that results in different amino acid sequences at their non-helical C-terminal regions. These sequences have been shown to interact with internal MHC amino acid residues and have a direct effect on crossbridge function and .alpha.-helical coiled coil formation (Martin, A. F. et al. (1997) Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 117:3-11).
Vertebrate smooth muscle contraction is dependent upon levels of cAMP and intracellular calcium ions ([Ca.sup.2+ ].sub.i). The sarcoplasmic reticulum (SR) serves as an intracellular store of [Ca.sup.2+ ].sub.i. Following stimulation by second messenger molecules, such as inositoltrisphosphate, [Ca.sup.2+ ].sub.i is briefly released from the SR into the surrounding cytoplasm. [Ca.sup.2+ ].sub.i binds to calmodulin (CaM), which activates CaM-dependent myosin light chain protein kinase (MLCK). MLCK then phosphorylates MLC. In relaxed muscle, myosin is prevented from interacting with actin by tropomyosin. Ca.sup.2+ binds to tropomyosin, causing a conformational change that leads to the release of actin. Phosphorylated MLC interacts with actin, forming actinomyosin, and initiates the contraction process. Muscle relaxation is brought about by active transport of Ca.sup.2+ into the SR by a calcium ATPase pump, and MLCK is inactivated by a cAMP-dependent protein kinase. Interactions between these molecules may be modulated by other proteins. In particular, telokin, a kinase-related protein encoded by the 3' region of the vertebrate smooth muscle MLCK gene, inhibits MLCK-dependent phosphorylation of MLC by modulating the oligomeric state of MLCK and its interaction with dephosphorylated myosin filaments (Nieznanski, K. and Sobieszek, A. (1997) Biochem. J. 322:65-71). Phosphorylation of caldesmon by casein kinase II has been shown to regulate the interactions between caldesmon and smooth muscle myosin and the ability of caldesmon to cross-link actin and myosin filaments (Sutherland, C. et al. (1994) J. Muscle Res. Cell. Motil. 15:440-456). Elevation of intracellular cGMP and activation of protein kinase G (PKG) produces relaxation of smooth muscle. Thirty one potential PKG substrates have been identified, including a protein complex containing proteins of 40, 33, 28, and 20 kDa (Li, H. et al. (1996) J. Vasc. Res. 33:99-110).
A Caenorhabditis elegans gene which encodes a protein similar to the coiled coil domain of MHC has recently been identified, but a biochemical or physiological role has yet to be established (Wilson, R. et al. (1994) Nature 368:32-38). Nasmyth, K. and Jansen, R. P. (1997; Curr. Opin. Cell Biol. 9:396-400) have suggested that proteins of the cytoskeleton, including unconventional myosins, play active roles in the segregation of differentiation factors and mRNA species during oogenesis and cell differentiation.
Numerous pathologies have been associated with mutations encoded within MHC isoforms, with differential expression of myosin heavy chain isoforms, and with differential activation of enzymes which chemically modify myosin or myosin-associated proteins (Abchee, A. and Marian, A. J. (1997) J. Investig. Med. 45:191-196). For example, elevated levels of PKC.beta.2 isoform associated with diabetes mellitus increase transcriptional activation of the fetal myosin heavy chain gene in adult myocardium. Together with increases in transcriptional activation of other genes, such as atrial natriuretic factor, c-fos, transforming growth factor, and collagens, this may lead to cardiomyopathy (Wakasai, H. et al. (1997) Proc. Natl. Acad. Sci. 94:9320-9325). Stromal nodules in benign prostatic hyperplasia (BPH) have morphological, cytoskeletal, and biochemical similarities to fetal prostate stroma supporting the idea of a reactivation of fetal processes in BPH (Bierhoff, E. et al. (1997) Prostate 31:234-240).
The discovery of two new human myosin heavy chain-like proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cell motility, reproductive, immunological, and neoplastic disorders.